Parkinson's disease (PD) is caused by the degeneration of midbrain dopamine (DA) producing cells and results in motor and non-motor impairments. Congenital and acquired forms of Parkinsonism, caused by inherited defects in DA synthesis or brain injury and disease, also produce profound immobility and disability. The hallmarks of Parkinsonism include bradykinesia, resting tremor, and postural instability that stem from a reduced supply of DA from the midbrain to the motor striatum, a brain structure required for motor control and cue-dependent learning. In turn, this loss of striatal DA prevents the normal spike-timing of the sparse population of tonically-active acetylcholine (ACh)-releasing interneurons (TANs) that provide the sole source of striatal ACh and are essential to many aspects of brain function. The resulting change in DA-ACh reciprocity contributes to the clinical symptoms and signs of Parkinsonism. Treatments that enhance DA availability or activate DA receptors incompletely restore motor function, become less successful over time, and are limited in efficacy due to induction of impulse control disorders and dyskinesias that are caused by neuroplasticity in TANs. Therefore, the precise targeting of ACh-releasing cells, while correcting for the decline in DA, would improve treatment and disability in Parkinsonism. TANs have been implicated in the pathophysiology of PD and treatment-dependent dyskinesias, but the mechanism whereby TANs participate in these movement disorders remains unclear. In this competing continuation, we will pursue an integrated series of molecular, electrophysiological, optical, and behavioral experiments in genetically engineered DA-deficient mice. Our goal is to show how DA deficiency promotes a long-lasting change in ACh release from TANs which contributes to motor features of Parkinsonism. We will test the hypotheses that 1) DA deficiency reduces autonomous firing in TANs by altering hyperpolarization-activated cation (HCN) channels that re-polarize the cell toward its resting membrane potential following each action potential; 2) DA replenishment provokes a paradoxical increase in TAN activity through DA and ACh auto-regulatory signal processing; and 3) DA deficiency produces plasticity in excitatory thalamostriatal inputs that contributes to the physiological and behavioral change. The central role of ACh in normal and abnormal movements has only recently been identified, despite clues from decades of empirical findings using anticholinergics. We now know that this is an important downstream response to DA deficiency. We will use innovative ways to measure transcribed mRNA and novel optical approaches to directly address alterations TAN function. These experiments provide a targeted molecular and circuit-level approach to cell-type specific effects which previous methods were not able to discern. By the end of this work, we will know the molecular and physiological mechanisms that regulate striatal ACh in DA deficiency and we will uncover pharmacological treatment targets that will improve DA-ACh reciprocity and motor function in children and adults with Parkinsonism.